About 12 years ago we discovered a new virus in our laboratory. The virus was present in the culture medium of an Aedes aegypti cell lie and was recognized because it caused fusion of Aedes albopictus cells. We called it CFA for cell fusing agent. We showed, at that time, that CFA was a 50 nm spherical, enveloped virus with a (+) strand RNA genome, that it replicated in the cytoplasm, and that it had three structural proteins. Although in these respects CFA resembled the togaviruses and flaviviruses, it shown no serological cross reaction with viruses in either of these two families. At present, the taxonomic status of CFA still remains unsettled. We propose now to extend our studies of CFA. There are two compelling reasons for undertaking these studies now. Within the past 6 months we have isolated by endpoint dilution, clones of CFA which replicate more quickly and produce yields 50-100 fold higher than our original strain. This will greatly facilitate biochemical studies. Secondly, since our original work was done, the field of recombinant DNA technology has emerged making possible new approaches to the study of viruses. In this proposal we describe experimental approaches which will help us learn more about how CFA replicates and about some of its biological properties. A major effort will be directed to the cloning and sequencing of the CFA genome. The information obtained will enable us to determine the number of open reading frames, to deduce the amino acid sequences of the viral proteins, and to see if there is any homology between CFA RNA and other viral RNAs. In direct studies on the CFA viral RNA we shall determine the nucleotide sequence of the 3' terminus of the RNA, in the process learning whether the RNA is polyadenylated, and we shall characterize the cap structure at the 5' terminus of the RNA. The CFA proteins will be sequenced at their N-termini so that their coding sequences can be aligned on the viral genome. Mutants of CFA will be selected, as we have done with Sindbis virus, which code for altered RNA capping and methylating enzymes. These will eventually be used for identifying the genes which code for these enzymes. We shall investigate in more detail the host range of CFA, testing its ability to replicate in other mosquito cell lines, in Drosophila and lepidopteran lines, and in selected vertebrate cells including a fathead minnow line. We shall test whether CFA can interfere with alpha and flaviviruses. Finally, we shall screen mosquitoes in New Jersey for viruses which resemble CFA. From these studies we shall 1) obtain a better understanding of how CFA replicates, 2) help settle the taxonomic status of CFA, and 3) gain insights into the evolution of small enveloped RNA viruses, including the arthropod-borne viruses which cause disease in man.